【作者】 丁飞；

【导师】 孙英； Steven R.Tannenbaum；

【作者基本信息】 中国农业大学 ， 农产品安全， 2013， 博士

【摘要】 长期以来,我国在巨大的人口压力下,农产品一直围绕着高产为主,从而大量施用各种农用化学品,包括杀虫剂、杀菌剂、除草剂、兽药、着色剂等。残留在农产品或环境中的农用化学品,通过直接或间接途径,最终进入人体蓄积,而大量农用化学品的毒副作用尚不明确。因此,“餐桌污染”问题越来越受到人们关注,农产品安全问题已成为全社会的焦点之一。人体内的生物大分子,例如蛋白质、核酸等是生命现象的物质基础,是参与人体内各种生物变化最重要的组分。蛋白质存在于一切细胞中,是构成人体的基本材料,且承担不同的生理功能,虽然核酸携带着人类的遗传信息,在人体的发育、生长、繁殖、遗传变异等生命过程中起极为重要的作用,但研究发现,核酸必须通过蛋白质来实现其生物学功能。所以,外源化合物例如农药、着色剂、兽药等,若干扰人体内蛋白质的正常生理活动过程,必然会导致人体某些器官机能紊乱,宏观表现为引发各种疾病。故而以蛋白质为考察对象,从分子层面研究农用化学品的毒性机理,有助于帮助人们评价这些农用化学品对人体的微观致毒机制,进而全面评估其毒性,为多种疾病的预防、诊断、治疗提供理论依据。本论文利用人体内含量最丰富、功能多样的血清白蛋白为模型,借助于多种生物物理方法从蛋白质水平进行了以下4个方面的创新性研究：(1)探讨了“低毒”杀虫剂吡虫啉、氯虫酰胺对模型蛋白HSA的毒性机理,并关注了手性农用化学品对非靶标生物的毒性；(2)选取某些典型食品着色剂为农用化学品污染物,通过对生物大分子的致毒机理分析,论证了以HSA为模型评价农用化学品毒性作用的有效性；(3)讨论了磺酰脲类除草剂对蛋白质毒性的微观信息与宏观生物活性之间的关联性,建立了基于构效关系的农用化学品致毒作用机制的预测模型；(4)揭示了人体内蛋白质很可能对某些食品添加剂的生理活性具有调节作用。论文分为以下6个部分：第一章：概述了农产品污染的现状、危害、影响因素,介绍了农用化学品的分类、毒性评价方法,总结了以蛋白质为靶标评估农用化学品毒性机理的研究手段及进展。第二章：研究了当前使用最广泛的农用化学品吡虫啉、氯虫酰胺、手性甲霜灵、氯霉素对模型蛋白质HSA的毒性作用,结果表明,4种农用化学品通过氢键、π-π、T-π、疏水作用与HSA中等强度络合,其中,HSA对(S)-甲霜灵的亲和力大于(R)-甲霜灵；除(R)-甲霜灵外,吡虫啉、氯虫酰胺、(S)-甲霜灵、氯霉素在HSA上只有一个结合位置(ⅡA亚域或ⅢA亚域),与核心氨基酸残基Phe-211、Trp-214、Arg-410、Tyr-411等产生非共价作用；4种农用化学品均能引起蛋白质多肽链伸展、蛋白质结构损伤。第三章：以HSA为靶标,考察了4种模型食品着色剂酸性红2、媒介红3、茜素氨羧络合剂、酚藏花红的毒理学机制,4种着色剂都能与靶标HSA形成复合物,因此,生色团荧光寿命几乎无变化,体系的Gibbs自由能变AG°<0,表明靶标-着色剂缔合过程是自发进行的放热反应；分子模型显示,食品着色剂与IIA亚域的Trp-214残基、IIIA亚域的Leu-407、Tyr-411、Phe-488、 Ser-489残基形成氢键、π-π、T-π、疏水作用,这些非共价作用导致多肽链解折叠、靶标蛋白空间构象改变,影响了其生理功能。第四章：利用权威环境物理化学和生物化学预测软件估算了44种磺酰脲类除草剂的环境参数,进行环境风险分级(Ⅰ、Ⅱ、Ⅲ级),从中选取了10个除草剂,利用多种生物物理学技术评价其对模型蛋白质HSA的毒性效应。光谱实验结果发现除草剂对HSA的毒性作用强弱与环境风险等级存在关联性,符合Ⅲ级>Ⅱ级>Ⅰ级的顺序；除草剂主要通过非共价作用,例如氢键、T-π、疏水作用缔合在模型蛋白HSA的ⅡA亚域(苄嘧磺隆位于Ⅲ亚域),引起蛋白质结构损伤,这个现象已由同步荧光、圆二色谱、三维荧光光谱所证实；氨基酸残基Lys-195、Trp-214、 Arg-222、Arg-257(ⅡA亚域)和Arg-410、Lys-414、Leu-491(ⅢA亚域)是除草剂-HSA轭合过程中关键氨基酸；构效关系分析可知,除草剂-HSA体系的亲和力与环境风险等级、辛醇-水分配系数密切相关,而且,除草剂分子结构中左、右两侧芳基、杂环电负性是决定其毒性强弱的核心因素。第五章：对比考察了天然产物黄烷酮类化合物橙皮苷、橙皮素的生理活性,荧光光谱显示黄烷酮类化合物-HSA的反应过程属于静态机理,这与时间分辨荧光测量结果相符,即形成了非荧光性复合物,生色团荧光寿命为5.32ns左右；橙皮苷、橙皮素通过氢键、T-π等非共价作用结合在HSA的ⅡA、ⅢA亚域,且ⅡA亚域对黄烷酮的亲和力大于ⅢA亚域,同时,络合过程引起HSA的α-螺旋结构含量降低,蛋白质的生理活性、功能发生改变；基于分子动力学模拟,可以看出黄烷酮位于ⅡA亚域时的稳定性强于ⅢA亚域,此外,橙皮苷在ⅡA、ⅢA亚域的稳定性均小于橙皮素,说明橙皮素-HSA复合物亲和力更大,这与分子模型结果非常一致。第六章：总结了本论文取得的主要研究结果,揭示了以人体内重要生物大分子HSA为模型进行毒性评价的有效性,并对后续研究进行了几点建议。更多还原

【Abstract】 Taking the long view, farm products in China always focused on high yielding because of the large pressure of potential population explosion; therefore various agrochemicals, such as insecticides, fungicides, herbicides, veterinary drugs and food colorants have been heavy used in almost every agricultural sector to keep a stable yields. Numerous studies have clearly demonstrated that agrochemicals residues in agricultural commodities or environments will finally be accumulated in critical human organs (e.g. heart, liver and kidney) through direct or indirect route. Worst of all, the toxicity and side effects of large quantity of agrochemicals still preserves opaque. Consequently,"fork contamination" attracted more and more attention and agricultural product safety has become a major concern of the contemporary society. Biomacromolecules such as protein and nucleic acids in the human body are the essential physical foundation of all forms of life, and they usually intervene in all kinds of life processes. Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of cells. It is the basic element of constructing human body and exhibit enormous diversity of biological function. Although nucleic acids stores and transmits genetic information from one generation to the next is a fundamental condition of life, scientific research found that the biological functions of nucleic acids must be achieved by different proteins. If the physiological activities of proteins were disturbed by several ligands such as pesticides, colorants and veterinary drugs, multiple organ dysfunction syndrome would be a direct consequence of the interference, and further various illnesses is the macroscopical representation of this event. Thus the study on toxicity mechanism of agrochemicals using protein as a model at the molecular level will help people see the microscopic poisoning mechanism when these agrochemicals entered human body. Furthermore, such study can also assess the overall toxic action of agrochemicals and provide the rationale for disease prevention, diagnosis and treatment.Given the above mentioned background, the main goal of the current thesis was to generate more comprehensive information about the toxicity of several typically agrochemicals by numerous biophysical techniques. Human serum albumin (HSA), which is the most abundant and multifunctional protein in the human body will be used as a model and the innovative results from four aspects were listed as follows:(1) The toxic action of "moderately" toxic insecticides imidacloprid and chlorantraniliprole to model HSA was discussed, and the toxicity of chiral agrochemicals to non-target organism was also attracted widely attention from here.(2) Through the poisonous mechanistic analyses of some properly selected food contaminant for biological macromolecule, this work demonstrated obviously that the validity of judging the toxic effect of agrochemicals using HSA as a suitable model.(3) The relationships between microscopic information and macroscopic biological activities of protein, which was created by the toxicity of sulfonylurea herbicides, were addressed in this thesis. Based on the structure-activity relationship analyses, the predicting modeling was proposed to forecast the poisonous mechanism of agrochemicals.(4) This research also brought out the biomacromolecules such as protein in the human body could probably have a significant impact on the physiological activities of some natural food additives.On the whole this thesis can be divided into the following six parts:Chapter1:In this section, the current situation, potential hazards and the influential factors of the contamination of agricultural products were reviewed. This part also introduced the classification of different agrochemicals and the methods of toxicological evaluation; moreover, the techniques and the recent progresses of toxic assessments for various agrochemicals using protein as a target were summarized herein.Chapter2:The assessment of the toxicity of several most widely used agrochemicals, i.e. imidacloprid, chlorantraniliprole,(R)-/(S)-metalaxyl and chloramphenicol for model macromolecule HSA is the subject of this chapter. Experiments show that the conjugation of four chemicals with HSA belongs to moderate strength, but the affinity of (S)-metalaxyl with protein is superior compared with that with (R)-metalaxyl. The primary forces between protein and ligands are hydrogen bonds, π-π, T-π and hydrophobic interactions; furthermore, agrochemicals are only situated within subdomain IIA or ⅢA except for (R)-metalaxyl, and then these compounds can make noncovalent interactions with some key amino acid residues such as Phe-211, Trp-214, Arg-410and Tyr-411. This event eventually led to the destabilization of the HSA spatial structure that is protein damage.Chapter3:The focus of this chapter is the inspection of the toxicology of model food colorants C.I. Acid Red2, C.I. Mordant Red3, alizarin complexone and phenosafranin using HSA as an appropriate target. The results display the complex of four food colorants to HSA is the formation of adduct, as a result the fluorescence lifetimes of fluorophore have barely changed in the process. According to the thermodynamics equation, the Gibbs free energy ΔG°<0, which intimates the formation of HSA-food colorants conjugation was an exothermic reaction. From molecular modeling outcomes, food colorants could form hydrogen bonds, π-π, T-π and hydrophobic interactions with several crucial amino acid residues such as Trp-214(subdomain IIA) or Leu-407, Tyr-411, Phe-488and Ser-489(subdomain IIIA). These noncovalent interactions caused the polypeptide chain of HSA partially unfolding with an alteration of protein conformation, which may be correlated with its physiological activity.Chapter4:In this chapter,44sulfonylurea herbicides were first divided into three environmental risk rating (Ⅰ,Ⅱ and Ⅲ) by employing some authoritative prediction software such as Estimation Program Interface (EPI) Suite, Toxicity Estimation Software Tool (TEST) and VEGA, and then10representative herbicides were selected and further assess the toxic effects for model protein HSA using multiple biophysical techniques. Spectroscopic experiments exhibited evidently some correlation exists between the toxicity of herbicides for HSA and the environmental risk rating of herbicides, and the correlativity is corresponding to the order:Ⅲ>Ⅱ>Ⅰ. The binding of herbicides to protein primarily took place in subdomain IIA whereas bensulfuron-methyl is located at subdomain IIIA, and the noncovalent interactions such as hydrogen bonds, T-π and hydrophobic interactions were found to be the principal acting force in stabilizing the complex. Molecular modeling indicated that some amino acid residues, e.g. Lys-195, Trp-214, Arg-222and Arg-257(subdomain IIA) and Arg-410, Lys-414and Leu-491(subdomain IIIA) plays an important role in the conjugation, and the intermolecular force between them engendered damage to protein, this corroborates synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectra that the spatial structure of HSA was changed. According to the structure-activity relationship, it may be observed that the affinity between herbicides and HSA is closely related to the environmental risk rating and the octanol-water partition coefficient; and furthermore, electronegativity of the aryl and heterocycle in herbicide molecule could highly probable exert a great impact on its toxicological properties.Chapter5:The discussion of this chapter will be limited to the comparative studies of the biological activities of natural flavanones hesperidin and hesperetin. The data of steady state fluorescence expounded the conjugation of flavanone with protein yielded quenching by a static mechanism, which substantiates time-resolved fluorescence measurements that non-fluorescent flavanone-HSA complex formation has a lifetime of5.32ns. Both subdomain IIA and IIIA were earmarked to possess high-affinity for flavanone, and the affinity of subdomain IIA with flavanone is greater than that of subdomain IIIA. Additionally, the primary forces between protein and flavanone are hydrogen bonds and T-π interactions, these intermolecular forces could induce the reduction of α-helix suggesting alterations of the structure and function of protein. In view of the molecular dynamics simulations, one may find that the stability of subdomain IIIA with flavanone is much lower than subdomain IIA. and the stableness of hesperetin in subdomain IIA and IIIA is superior, compared with hesperidin; this coincides perfectly with molecular modeling results that the strength of hesperetin with HSA is bigger than hesperidin.Chapter6:This section is a primary conclusion of the preceding chapters, uncovering the credibility of the toxicological risk assessment by exploiting several fundamental biopolymers in the human body such as HSA as a valuable model. Finally, some suggestions for future research are also proposed in this part.更多还原